Isolation Of Plant Pigments From Spinach By Thin Layer Chromatography

Isolation of Plant Pigments from Spinach by Thin Layer Chromatography The leaves of plants contain a number of colored pigments generally falling into two categories, chlorophylls and carotenoids. Chlorophylls a and b are the pigments that make plants look green. These highly conjugated compounds capture the (nongreen) light energy used in photosynthesis.

Carotenoids are part of a larger collection of plant derived compounds called terpenes. These naturally occurring compounds contain 10, 15, 20, 25, 30 and 40 carbon atoms which suggest that there is a compound with five carbon atoms that serves as their building block. Their structures are consistent with the assumption that they were made by joining together isoprene units, usually in a "head to tail" fashion. Isoprene is the common name for 2-methyl-1,3-butadiene. The branched end is the "head" and the unbranched is the "tail". That isoprene units are linked in a head to tail fashion to form terpenes is known as the isoprene rule. Carotenoids are tetraterpenes (eight isoprene units). Lycopene, the compound responsible for the red coloring of tomatoes and watermelon, and b -carotene, the compound that causes carrots and apricots to be orange, are examples of carotenoids. b -Carotene is also the coloring agent used in margarine. When ingested b -Carotene is cleaved to form two molecules of vitamin A and is the major dietary source of the vitamin. Vitamin A, also called retinol, plays an important role in vision. Spinach leaves contain chlorophyll a and b and b -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls which are oxidized versions of carotenes and pheophytins which look like chlorophyll except that the magnesium ion Mg++ has been replaced by two hydrogen ions 2H+. In this experiment we will isolate and separate the spinach pigments using differences in polarity to effect the separation. Since the different components are colored differently, we can follow this separation visually. The structures of the major components are given below. Notice that since b -carotene is a hydrocarbon it is very nonpolar. Both

chlorophylls contain C--O and C--N bonds which are polar and also contain magnesium bonded to nitrogen which is such a polar bond it is almost ionic. Both chlorophylls are much more polar than b -carotene. If you look carefully you can see that the two chlorophylls differ only in one spot. Chlorophyll a has a methyl group (--CH3) in a position where chlorophyll b has an aldehyde (--CHO). This makes chlorophyll b slightly more polar than chlorophyll a. After we isolate the pigment mixture from the leaves in a hexane solution we will use the difference in polarity to separate the various pigments using column chromatography. We will analyze the original extract and the pigment fractions using thin layer chromatography, which also separates based on polarity.

Chlorophyll a (Blue-green, polar) C55H72MgN4O5

M. W. 893.5026

Chlorophyll b (Green, polar) C55H70MgN4O6 M. W. 907.4862

beta-Carotene--yellow, nonpolar C40H56 M.W. 536.8824